开题报告内容：（包括拟研究或解决的问题、采用的研究手段及文献综述，不少于2000字）
一、课题背景 (Introduction)
The Na - and K -dependent adenosine triphosphatase (Na/K-ATPase, or sodium pump), was discovered by Skou in 1957[1]. Na/K-ATPase consists of two noncovalently linked subunits, alpha; and beta; subunits. The alpha; subunit has four isoforms, alpha;1 is ubiquitously expressed in all cell types while alpha;2, alpha;3 and alpha;4 are expressed in a tissue-specific manner[2, 3, 4, 5]. Cardiotonic steriods (CTS), including plant-derived digitalis drugs like ouabain and digoxin, and vertebrate-derived aglycones like bufalin and marinobufagenin (MBG)[6, 7], are specific inhibitors of Na/K-ATPase.The positive inotropic effect of CTS are due to the inhibition of NKA, and resulted in increases in Ca2 oscillation[8]. Like other inotropes, the FDA approved CTS digoxin produces impressive hemodynamic effects in chronic heart failure (CHF) patients. However, it distinguishes itself from other pure inotrope drugs because it does not increase mortality of CHF patients, most likely due in part to the recently appreciated signaling through cardiac Na/K-ATPase[9, 10]. Several endogenous CTS, including ouabain and marinobufagenin, have been identified, and they do play an important role in the regulation of cardiac structure and function[11, 12, 13].
Apart from the pumpting function, recent studies have also confirmed that Na/K-ATPase acts as a signal transducer. This ligand bind can activate the NKA-associated Src and result in the generation and amplification of the ligand-binding signal to increase in the protein tyrosine phosphorylation. For instance, CTS activation of Na/K-ATPase/Src receptor complex is capable of transactivating the epidermal growth factor receptor (EGFR) and the subsequent stimulation of Ras/Raf/MEK/ERK1/2 [14, 15]; Whatrsquo;s more, the actication of Src can result in the stimulation of phospholipase (PLC-ᵞ) , thus activating protein kinase C ε isozymes (PKCε)[16]. CTS activation of NKA-mediated intracellular signaling represents a novel mechanism for the modulation of a variety of cell processes including cell growth and apoptosis, and is involved in both normal cardiac physiology[17] and diseases such as ventricular hypertrophy[18] and fibrosis[19, 20].
In the human heart, alpha;2 and alpha;3 are differentially expressed in developmental and pathological conditions. Modulations including hypertrophy and adverse cardiac remodeling have been report in both human biopsies and animal models for decades, but limited knowledge, if any, has been garnered on the causative role of Na/K-ATPase alpha; isoform remodeling in Hypertrophic cardiomyopathy (HCM) or their potential use as novel biomarkers /therapeutic targets. Clearly, a major hurdle to progress in this line of research has been the absence of an adequate cellular model, owing mostly to the limited lifetime of cardiac myocytes in expression since the adultrodent heart completely lacks alpha;3.
In the past few years, human induced pluripotent stem cells (hiPSCs) have emerged as a promising source of phenotypically authentic cardiomyocytes and provided a human-based in vitro cardiomyocyte model system for us to study the role of cardiac Na/K-ATPase alpha;-isoforms in CMH. Because hiPSCs are stable in long-time culture and show relative ease of genetic manipulation compared to adult primary cardiomyocytes, it will allow us to selectively alter Na/K-ATPase isoforms and establish a causative role in the initiation and/or the progression of CMH in a human cardiac myocyte.
- 要解决的问题 (Problems to be solved)
At the molecular level, we suggest that receptor Na/K-ATPase regulates protein/protein interaction in a conformation-dependent manner. The capability of normal conformation transition is essential for both pumping and signaling functions of Na/K-ATPase[21]. CTS share a steroid core, a varying number of substituents, and a five- or six-membered lactone, which has specific influence on the binding properties to Na/K-ATPase[22]. While all CTS bind to the E2P state of Na/K-ATPase, they do induce different Na/K-ATPase structural changes. We suggest that the structural rearrangement trigged by individual CTS may affect different protein–protein interaction modes within the intracellular signal transduction networks[23, 24]. As a consequence, the allosteric control by individual CTS may stabilize Na/K-ATPase in a unique conformation, leading to biased activation of a specific signaling pathway as exampled by biased GPCR ligands[25], Based on our preliminary studies of ouabain, digitoxigenin and somalin, we propose that Na/K-ATPase works as signaling receptor by direct protein/protein interaction with effectors in a conformation-dependent manner. As such, different CTS, like GPCR ligands, could trigger individual CTS-specific protein/protein interactions, resulting in biased signaling responses. Because recent studies have not only demonstrated the existence of biased signaling, but also indicated that the biased beta;-blocker carvedilol may be a better drug than the balanced blockers[26-30], we further suggest that understanding of biased signaling may render a novel CTS a better therapeutic index than the only FDA-approved CTS digoxin.
Accordingly, the following aims are proposed to test the hypothesis that structurally divergent CTS can produce a spectrum of biased signaling action with relevance in human cardiac myocytes .
Aim1: To test the hypothesis that CTS can activate biased signaling pathways from receptor Na/K-ATPase/Src complex, we will do detailed dose-response curves of digoxin, digitoxigenin, MIIR-B1, MIIR-B2, MIIR-B4, MIIR- B5 on ERK and PKC activation as well as the endocytosis of NKA, and then calculate EC50 and biased factor using ouabain as a reference.
Aim2: To assess human and cardiac relevance of our new findings, we will verify key findings in cardiac myocytes generated from human induced pluripotent stem (iPS) cells.
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